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2 phenotype
Clinica Chimica Acta 348 (2004) 35 – 40 www.elsevier.com/locate/clinchim
Detailed analysis of serum lipids and lipoproteins from Japanese type III hyperlipoproteinemia with apolipoprotein E2/2 phenotype Yasuhiro Todo, Junji Kobayashi *, Toshinori Higashikata, Masaaki Kawashiri, Atsushi Nohara, Akihiro Inazu, Junji Koizumi, Hiroshi Mabuchi Department of Internal Medicine, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan Received 8 March 2004; received in revised form 26 April 2004; accepted 26 April 2004
Abstract Background: To clarify a detailed profile of serum lipids, lipoproteins and apolipoproteins (apo) in type III hyperlipoproteinemia (HLP) with apolipoprotein E (apo E) phenotype 2/2. Methods: Nineteen consecutive Japanese type III HLP (9 men, 10 women) were studied. All had hypertriglyceridemia and 74% showed hypercholesterolemia. Results: The degree of hyperlipidemia [total cholesterol (TC) 8.1 F 3.2 mmol/l, triglycerides (TG) 5.2 F 2.9 mmol/l] was milder than that in type III HLP in western countries. Lipoprotein fractions analyzed by ultracentrifugation showed that very low density lipoprotein cholesterol (VLDL-C) concentrations were considerably increased and that intermediate density lipoprotein cholesterol (IDL-C) concentrations were also increased, whereas low-density lipoprotein cholesterol (LDL-C) concentrations were low. Serum apo A-I, A-II and B concentrations were not increased, while apo C-II, C-III and E concentrations were considerably increased. However, the increase of apo E concentrations in the study subjects was far more pronounced than that of apo C-III, causing the ratio of apo E/C-III to be considerably higher than hyperlipidemia with other apo E phenotypes. Conclusion: By using this index apo E/C-III, it is possible to segregate type III HLP with apo E2/2 phenotype from other types of hyperlipidemia. D 2004 Elsevier B.V. All rights reserved. Keywords: Apo E/C-III ratio; Ultracentrifugation; VLDL-C/TG ratio
1. Introduction Apolipoprotein E (apo E) is a 299-amino acid protein and is a constituent of chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and a subclass of high-density lipoprotein (HDL) [1 –3]. Three major apo E isoforms are * Corresponding author. Tel.: +81-76-265-2268; fax: +81-76234-4246. E-mail address: [email protected] (J. Kobayashi). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.04.018
coded by three alleles at the apo E locus, designated E2, E3 and E4, giving rise to six common phenotypes. The most common isoform, E3, is characterized by a cysteine at amino acid residue 112 and an arginine at residue 158. The E2 isoform has a cysteine at residue 112 and 158 which binds poorly to hepatic lipoprotein receptors [3,4], and that in general population, the E2 allele is consistently associated with increased concentration of triglycerides (TG) and apo E compared with the E3 allele [5– 8]. Homozygote of apo E2, coupled with
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Y. Todo et al. / Clinica Chimica Acta 348 (2004) 35–40
environmental factors such as diabetes, overeating, thyroid function, etc., is known to contribute to the formation of type III hyperlipoproteinemia (HLP) [3,9 – 11]. Type III hyperlipoproteinemia is one of the five familial HLP defined by Fredrickson et al. [12] in the 1960s; the biochemical and clinical features of which are varied and not all of the abnormalities are present in every patient [3,13]. Plasma total cholesterol (TC) and triglycerides (TG) are increased to approximately equal concentrations, usually >7.80 mmol/l. So far, there are very few reports on plasma lipid and lipoprotein profiles for type III HLP with apo E2/ 2 phenotype in Japanese population due in part to its rarity [14,15]. We analyzed detailed profiles of serum lipids and lipoproteins in 19 unrelated Japanese type III HLP with apo E2/2 phenotype.
2. Materials and methods
(Daiichi Chemical Tokyo, Japan). Serum TC concentrations z 220 mg/dl is defined as hypercholesterolemia and serum TG concentrations z 150 mg/dl as hypertriglyceridemia [16]. Lipoprotein fractions in sera were separated by serial ultracentrifugation at different densities into VLDL (d < 1.006), IDL (d = 1.006 to 1.019), lowdensity lipoprotein (LDL; d = 1.019 to 1.063) and HDL (d>1.063) according to the method by Havel et al. [17]. The serum remnant-like particle cholesterol (RLPC) concentration was measured using an immunoaffinity mixed gel containing anti-apolipoprotein A-I (apo A-I) and anti-apo B monoclonal antibody, following the method reported by Nakajima et al. [18]. 2.3. Isoform analyzed by isoelectric focusing (IEF) To determine the apo E phenotype for the study subjects, IEF was performed in polyacrylamide gels essentially as described previously [19,20].
2.1. Patients 2.4. Statistical analysis All 19 consecutive type III HLP patients (9 men, 10 women) were Japanese and were recruited from our clinics and affiliated hospitals. Age and body mass index (BMI) of the subjects were 52.5 F 15.9 years old and 23.9 F 2.9 kg/m2, respectively. The diagnosis of type III HLP was performed based on the standard criteria previously reported [15]. Individuals with plasma glucose (PG) z 7.0 mmol/l after overnight fast or those with z 11.1 mmol/l at 2 h after 75-g oral glucose administration documented on >1 occasion were diagnosed as diabetes mellitus (DM). Coronary heart disease (CHD) was diagnosed according to the following criteria: (1) a history of myocardial infarction; (2) a history of angina pectoris together with a positive exercise test result and/or an abnormal coronary angiogram; and (3) a history of coronary artery bypass grafting. Blood sample was obtained after overnight fasting. 2.2. Serum lipoprotein analysis Serum TC, TG and HDL cholesterol (HDL-C) concentrations were determined by standard enzymatic methods. Serum apolipoprotein (apo) concentrations were determined using an immunoturbidimetric assay
All values are expressed as the mean F S.D. StatView-J 5.0 software was used for all statistical analyses. Statistical significance between two groups was determined by unpaired Student’s t test at P < 0.05. The Mann – Whitney test was used to detect the differences between the groups if data did not distribute normally.
3. Results 3.1. Clinical characteristics of the study subjects Among the study subjects, seven patients (36.8%) had obesity and five patients (26.3%) type 2 diabetes mellitus (DM). There were three patients (15.8%) who had coronary heart disease (CHD) and one (5.3%) with atherosclerotic obliterance (ASO). None of the subject had overt cerebral vascular disease. Xanthoma striata palmaris was observed in three patients (15.8%) and Achilles tendon’s xanthoma in three patients (15.8%). Serum lipid, lipoprotein, apolipoprotein, Lp (a), RLP-C concentrations and VLDL cholesterol (VLDL-C)/TG ratio in the study subjects
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are shown in Table 1. Average values for serum TC and TG were 8.1 and 5.2 mmol/l, respectively. All of the patients had hypertriglyceridemia whereas 14 patients (74%) had hypercholesterolemia. Average concentration of HDL-C was within normal limits. Average concentrations of serum apo A-I, A-II and B were within normal limits whereas apo C-II, C-III and E concentrations were markedly increased. In particular, average concentrations for apo E were as high as threefold higher than the upper limits of normal range whereas apo C-II and C-III concentrations were at most twofold higher. 3.2. Apo E/C-III ratio in hyperlipidemic individuals with apo E2/2, E3/2, E3/3, E4/3, E4/2 and E4/4 Fig. 1 shows the comparison of apo E/C-III ratio in individuals with apo E2/2, E3/2, E3/3, E4/3, E4/2 and E4/4. Apo E/C-III ratio from hyperlipidemia with apo E2/2 phenotype was considerably higher than that from other apo E phenotypes, while that from other phenotypes showed almost similar concentrations. Next, we investigated how serum lipids affect this index versus the absolute values of serum apo E concentrations. Apo E values were closely associated with both serum TC and TG concentrations, whereas apo E/C-III ratio had no association with either of these lipids (Table 2). This observation suggests that this index, unlike apo E value, could be a reliable Table 1 Serum lipid, lipoprotein and apolipoproteins values in study subjects (mean F S.D.)
Fig. 1. The comparison of apo E/C-III ratio between hyperlipidemic individuals with various apo E phenotypes. Values are mean F S.D. Hyperlipidemic individuals were used with apo E3/2 (n = 6) [TC 6.70 F 1.35 mmol/l, TG 4.53 F 3.55 mmol/l], E3/3 (n = 19) [TC 6.62 F 1.69 mmol/l, TG 4.27 F 5.84 mmol/l], E4/3 (n = 10) [TC 5.79 F 1.25 mmol/l, TG 5.18 F 3.63 mmol/l], E4/2 (n = 8) [TC 6.73 F 1.17 mmol/l, TG 2.42 F 1.98 mmol/l] and E4/4 (n = 9) [TC 6.70 F 1.38 mmol/l, TG 1.33 F 0.55 mmol/l] besides the study subjects with apo E2/2. ***p < 0.0001 vs. hyperlipidemic individuals with all of the other apo E phenotypes.
marker for screening subjects with apo E2/2 phenotype regardless of serum lipids. To determine the accuracy of apo E/C-III ratio for diagnosing type III HLP, we conducted ROC plot analysis (Table 3). The ROC analysis showed that sensitivities almost equaled specificities at apo E/C-III between 0.70 and 0.75 for identifying type III HLP with apo E2/2. 3.3. Gender differences in the parameters on lipoprotein metabolism between men and women
Normal range N Age, year BMI, kg/m2 TC, mmol/l TG, mmol/l HDL-C, mmol/l Apo A-I, mg/dl Apo A-II, mg/dl Apo B, mg/dl Apo C-II, mg/dl Apo C-III, mg/dl Apo E, mg/dl Apo E/C-III, mg/dl Lp(a), mg/dl RLP-C, mg/dl VLDL-C/TG
19 52.5 F 15.9 23.9 F 2.9 8.1 F 3.2 5.2 F 2.9 1.1 F 0.2 133 F 24.7 37.5 F 8.0 120 F 63.8 11.1 F 4.9 20.4 F 9.3 18.6 F 7.5 0.911 9.9 F 5.0 n = 7 19.6 F 8.1 n = 7 0.32 F 0.07
We also analyzed the differences in the parameters on lipoprotein metabolism between men and 3.4 – 5.7 0.4 – 1.7 0.9 – 2.2 97 – 167 24 – 47 52 – 130 1.5 – 5.1 4.7 – 11.9 2.1 – 6.0 34.6> 7.5>
Table 2 Relationship of TC or TG with apo E or E/C-III R
p
To TC Apo E 0.862 < 0.0001 Apo E/C-III 0.091 NS - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - -- - - - - To TG Apo E 0.828 < 0.0001 Apo E/C-III 0.125 NS TC, total cholesterol; TG, triglycerides.
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Table 3 ROC analysis: ranges of cutoff lines and their corresponding specificities and sensitivities for apo E/C-III
RLP-C concentrations and VLDL-C/TG ratio (data not shown).
Cutoff line
Specificities
1-Specificities
Sensitivities
1.55 1.50 1.45 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05 1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25
1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.96 0.96 0.96 0.96 0.96 0.96 0.92 0.92 0.88 0.85 0.85 0.77 0.67 0.58 0.40 0.31 0.15 0.06 0.02 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.04 0.04 0.04 0.04 0.04 0.04 0.08 0.08 0.12 0.15 0.15 0.23 0.33 0.42 0.60 0.69 0.85 0.94 0.98 1.00
0.00 0.11 0.11 0.11 0.11 0.11 0.22 0.28 0.33 0.33 0.33 0.39 0.50 0.56 0.61 0.67 0.83 0.89 0.94 0.94 0.94 0.94 0.94 1.00 1.00 1.00 1.00
3.4. Profiles of lipoprotein fractions analyzed by ultracentrifugation from the study subjects Table 4 shows the profiles of lipoprotein fractions analyzed by ultracentrifugation from the study subjects. VLDL-C concentrations were considerably increased. IDL cholesterol (IDL-C) concentrations were also increased whereas LDL cholesterol (LDL-C) concentrations were low. VLDL-TG, IDL TG, LDL-TG and HDL-TG concentrations were increased. HDL-C was within normal limits while HDL-TG concentrations were increased in type III HLP. Again, there was no significant difference between men and women for the profiles of lipoprotein fractions. The VLDL-C/TG ratio, which is regarded as a useful index for diagnosing type III HLP, was 0.32 F 0.07 in our study subjects.
4. Discussion
women. There was no statistically significant gender differences for TC, TG, HDL-C, apo A-I, apo A-II, apo B, apo C-II, apo C-III, apo E, Lp(a),
The main findings of the present study on detailed analysis of serum lipids and lipoproteins from 19 unrelated Japanese type III HLP with apo E2/2 phenotype are as follows: (1) all of the study subjects had increased serum TG, while 74% of the subjects had hypercholesterolemia; and (2) the increase of apo E concentration was far more pronounced than that of
Table 4 Ultracentrifugation analysis of serum lipoprotein values in men and women (mean F S.D.)
N Cholesterol VLDL IDL LDL HDL Triglycerides VLDL IDL LDL HDL
Total
Men
Women
Normal controls
19 7.8 F 3.1*** 3.7 F 2.1*** 1.1 F 0.5** 1.4 F 0.8*** 1.1 F 0.3 4.5 F 2.5*** 3.3 F 1.9*** 0.4 F 0.2*** 0.3 F 0.1** 0.3 F 0.1***
9 7.1 F 2.5*** 3.6 F 2.3*** 0.9 F 0.4*** 1.4 F 0.9*** 0.9 F 0.3* 4.5 F 2.6*** 3.6 F 2.4*** 0.3 F 0.2*** 0.3 F 0.1* 0.3 F 0.1***
10 8.5 F 3.6*** 3.8 F 2.0*** 1.2 F 0.5*** 1.4 F 0.7*** 1.2 F 0.3 4.5 F 2.5*** 3.0 F 1.3*** 0.4 F 0.3*** 0.3 F 0.1* 0.3 F 0.03***
20 4.8 F 0.7 0.4 F 0.2 0.2 F 0.1 2.5 F 0.6 1.2 F 0.3 1.0 F 0.4 0.5 F 0.3 0.1 F 0.03 0.2 F 0.1 0.2 F 0.03
VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. * p < 0.05 vs. control. ** p < 0.01 vs. control. *** p < 0.001 vs. control.
Y. Todo et al. / Clinica Chimica Acta 348 (2004) 35–40
apo C-III, causing the ratio of apo E/C-III to be considerably higher than individuals with other apo E phenotype. The findings that average concentrations for serum TC and TG in our study subjects were lower than those from type III HLP in western countries were in line with the observation in other Japanese group [15]. It is known that hyperlipidemia with apo E2/2 has marked increase of apo E concentrations [21 –25]. However, we presume that high serum apo E concentration per se is not sufficient to segregate apo E2/2 subjects in view of the fact that apo E concentration depends highly on serum TC and TG concentrations (Table 2). Indeed, in the U.S., the proposed diagnostic concentrations of plasma apo E concentrations for type III HLP is controversial [21,24]. In contrast, the ratio of apo E/ C-III is not affected by TC and TG concentrations, which is close to 0.9 in individuals with apo E2/2 (Fig. 1; Table 2). Thus, we suggest that this index is a useful marker for screening type III HLP with apo E2/2 phenotype from other type of HLP. We assume it is possible to segregate type III HLP with apo E2/2 regardless of the degree of hyperlipidemia. Another marker for diagnosing type III HLP might be a VLDLC/TG ratio of >0.30 [9]. In our study, the average value for this ratio was 0.32 F 0.07 which is close to the report by Janus et al. [23] and is higher than the finding by another Japanese group [15]. Several other markers have been shown useful for segregating HLP with apo E2/2 phenotypes [26,27]. Sakai et al. [26] showed that apo B-48/TG was almost equal to 0.15 in HLP with apo E2/2 phenotypes, whereas in all of other lipid phenotype, this ratio was V 0.064. Wang et al. [27] proposed that RLP-C/TG is a useful marker for segregating type III HLP from other type of HLP. We presume that these markers are also very useful for the diagnosis of type III HLP. However, a big advantage of our marker over these reports is that we do not have to use values such as apo B48 and RLP-C which are not routine available. The ultracentrifugation analysis of serum lipoproteins showed that the HDL-C was within normal limits while HDL-TG concentrations were increased in type III HLP, suggesting that HDL particles are relatively TG-rich in type III HLP. Subjects with type III HLP occasionally have xanthoma striata palmaris and Achilles tendon’s xanthoma, which were found 15.8% and 15.8%, respectively, in our study subjects. One of the clinically most
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important complications of type III HLP is known to be coronary heart disease (CHD) which was shown in 15.8% of our study subjects, whereas other Japanese group has reported its frequency to be 37.5%. The precise factors causing this discrepancy in CHD frequency between our study and other group remains to be clarified; but it may be related to the fact that in the present study, 37% of the patients were obese and 26% had type 2 diabetes, while their study subjects consisted in 75% obesity and 43.5% DM. In conclusion, we conducted a detailed analysis of serum lipids and lipoproteins in Japanese type III HLP with apo E 2/2 and therefore propose that apo E/C-III is a useful index for screening HLP with apo E 2/2 phenotype. Acknowledgements We thank Ms. Yoshida and Ms. Mizuno for their excellent technique for analyzing serum lipoprotein profiles of the study subjects. References [1] Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988;240: 622 – 30. [2] Mahley RW, Rall Jr SC. Apolipoprotein E: far more than a lipid transporter protein. Annu Rev Genomics Hum Genet 2000;01:507 – 37. [3] Mahley RW, Rall Jr SC. Type III hyperlipoproteinemia: the role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B, editors. The metabolic and molecular bases of inherited disease, vol. 2. New York: McGraw-Hill; 2001. p. 2835 – 62. [4] Weisgraber KH, Innerarity TL, Mahley RW. Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine – arginine interchange at a single site. J Biol Chem 1982;257:2518 – 21. [5] Utermann G, Pruin N, Steinmetz A. Polymorphism of apolipoprotein E: III. Effect of a single polymorphic gene locus on plasma lipid concentrations in man. Clin Genet 1979;15: 63 – 72. [6] Utermann G, Vogelberg KH, Steinmetz A, Schoenborn W, Pruin N, Jaeschke M, et al. Polymorphism of apolipoprotein E: II. Genetics of hyperlipoproteinemia type III. Clin Genet 1979;15:37 – 62. [7] Utermann G, Hees M, Steinmetz A. Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinaemia in man. Nature 1977;269:604 – 7.
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[8] Hagberg JM, Wilund KR, Ferrell RE. APO E gene and geneenvironment effects on plasma lipoprotein – lipid concentrations. Physiol Genomics 2000;4:101 – 8. [9] Morganroth J, Levy RI, Fredrickson DS. The biochemical, clinical, and genetic features of type III hyperlipoproteinemia. Ann Intern Med 1975;82:158 – 74. [10] Feussner G, Wagner A, Kohl B, Ziegler R. Clinical features of typeIII hyperlipoproteinemia: analysis of 64 patients. Clin Investig 1993;71:362 – 6. [11] Dyerberg J. Type 3 hyperlipoproteinemia with low plasma thyroxine binding globulin. Metabolism 1969;18:50 – 7. [12] Fredrickson DS, Levy RI, Lees RS. Fat transport in lipoproteins—an integrated approach to mechanisms and disorders. N Eng J Med 1967;276:34 – 44, 94 – 103, 148 – 56, 215 – 25, 273 – 81. [13] Mahley RW, Huang Y, Rall SC. Patheogenesis of type III hyperlipoproteinemia: questions, quandaries, and paradoxes. J Lipid Res 1999;40:1933 – 49. [14] Eto M, Watanabe K, Ishii K. A racial difference in apolipoprotein E allele frequencies between the Japanese and Caucasian populations. Clin Genet 1986;30:422 – 7. [15] Eto M, Nakata M, Iwashima Y, Watanabe K, Ikoda A, Kaka K. Type III hyperlipoproteinemia with apolipoprotein E2/2 genotype in Japan. Clin Genet 2002;61:416 – 22. [16] Hata Y, Mabuchi H, Saito Y, et al. Report of the Japan Atherosclerosis Society (JAS) guideline for diagnosis and treatment of hyperlipidemia in Japanese adults. J Atheroscler Thromb 2002;9:1 – 27. [17] Havel RL, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345 – 53. [18] Nakajima K, Saito T, Tamura A, Suzuki M, Nakano T, Adachi M, et al. Cholesterol in remnant like lipoproteins in human serum using monoclonal anti-apoB-100 and anti-apo A-I immunoaffinity mixed gels. Clin Chim Acta 1993;223:53 – 71.
[19] Matsunaga A, Sasaki J, Moriyama K, Arakawa F, Takada Y, Nishi K, et al. Population frequency of apo E5 (Glu3 ! Lys) and E7 (Glu244 ! Lys) variants in western Japan. Clin Genet 1995;48:93 – 9. [20] Moriyama K, Sasaki J, Matsunaga A, Arakawa F, Takada Y, Araki K, et al. Apolipoprotein E1 Lys 146 Glu with type III hyperlipoproteinemia. Biochem Biophys Acta 1992;1128: 58 – 64. [21] Brewer Jr HB, Zech LA, Gregg RE, Schwartz D, Schaefer EJ. NIH conference. Type III hyperlipoproteinemia: diagnosis, molecular defects, pathology, and treatment. Ann Intern Med 1983;98:623 – 40. [22] Feussner G, Feusssner V, Hoffmann MM, Lohrmann J, Wieland H, Marz W. Molecular basis of type III hyperlipoproteinemia in Germany. Human Mutat 1998;11:417 – 23. [23] Janus ED, Grant S, Lincott VJ, Wardell MR. Apolipoprotein E phenotypes in hyperlipidemic patients and their implications for treatment. Atherosclerosis 1985;57:249 – 66. [24] Kushwaha RS, Hazzard WR, Wahl PW, Hoover JJ. Type III hyperlipoproteinemia: diagnosis in whole plasma by apolipoprotein-E immunoassay. Ann Intern Med 1977;87: 509 – 16. [25] Havel RJ, Kane JP. Primary dysbetalipoproteinemia: predominance of a specific apoprotein species in triglyceride-rich lipoproteins. Proc Natl Acad Sci U S A 1973;70:2015 – 9. [26] Sakai N, Uchida Y, Ohashi K, Hibuse T, Saika Y, Tomari Y, et al. Measurement of fasting serum apoB-48 concentrations in normolipidemic and hyperlipidemic subjects by ELISA. J Lipid Res 2003;44:1256 – 62. [27] Wang T, Nakajima K, Leary ET, Warnick GR, Cohn JS, Hopkins PN, et al. Ratio of remnant-like particle-cholesterol to serum total triglycerides is an effective alternative to ultracentrifugal and electrophoretic methods in the diagnosis of familial type III hyperlipoproteinemia. Clin Chem 1999;45: 1981 – 7.
Detailed analysis of serum lipids and lipoproteins from Japanese type III hyperlipoproteinemia with apolipoprotein E2/2 phenotype Yasuhiro Todo, Junji Kobayashi *, Toshinori Higashikata, Masaaki Kawashiri, Atsushi Nohara, Akihiro Inazu, Junji Koizumi, Hiroshi Mabuchi Department of Internal Medicine, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan Received 8 March 2004; received in revised form 26 April 2004; accepted 26 April 2004
Abstract Background: To clarify a detailed profile of serum lipids, lipoproteins and apolipoproteins (apo) in type III hyperlipoproteinemia (HLP) with apolipoprotein E (apo E) phenotype 2/2. Methods: Nineteen consecutive Japanese type III HLP (9 men, 10 women) were studied. All had hypertriglyceridemia and 74% showed hypercholesterolemia. Results: The degree of hyperlipidemia [total cholesterol (TC) 8.1 F 3.2 mmol/l, triglycerides (TG) 5.2 F 2.9 mmol/l] was milder than that in type III HLP in western countries. Lipoprotein fractions analyzed by ultracentrifugation showed that very low density lipoprotein cholesterol (VLDL-C) concentrations were considerably increased and that intermediate density lipoprotein cholesterol (IDL-C) concentrations were also increased, whereas low-density lipoprotein cholesterol (LDL-C) concentrations were low. Serum apo A-I, A-II and B concentrations were not increased, while apo C-II, C-III and E concentrations were considerably increased. However, the increase of apo E concentrations in the study subjects was far more pronounced than that of apo C-III, causing the ratio of apo E/C-III to be considerably higher than hyperlipidemia with other apo E phenotypes. Conclusion: By using this index apo E/C-III, it is possible to segregate type III HLP with apo E2/2 phenotype from other types of hyperlipidemia. D 2004 Elsevier B.V. All rights reserved. Keywords: Apo E/C-III ratio; Ultracentrifugation; VLDL-C/TG ratio
1. Introduction Apolipoprotein E (apo E) is a 299-amino acid protein and is a constituent of chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and a subclass of high-density lipoprotein (HDL) [1 –3]. Three major apo E isoforms are * Corresponding author. Tel.: +81-76-265-2268; fax: +81-76234-4246. E-mail address: [email protected] (J. Kobayashi). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.04.018
coded by three alleles at the apo E locus, designated E2, E3 and E4, giving rise to six common phenotypes. The most common isoform, E3, is characterized by a cysteine at amino acid residue 112 and an arginine at residue 158. The E2 isoform has a cysteine at residue 112 and 158 which binds poorly to hepatic lipoprotein receptors [3,4], and that in general population, the E2 allele is consistently associated with increased concentration of triglycerides (TG) and apo E compared with the E3 allele [5– 8]. Homozygote of apo E2, coupled with
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Y. Todo et al. / Clinica Chimica Acta 348 (2004) 35–40
environmental factors such as diabetes, overeating, thyroid function, etc., is known to contribute to the formation of type III hyperlipoproteinemia (HLP) [3,9 – 11]. Type III hyperlipoproteinemia is one of the five familial HLP defined by Fredrickson et al. [12] in the 1960s; the biochemical and clinical features of which are varied and not all of the abnormalities are present in every patient [3,13]. Plasma total cholesterol (TC) and triglycerides (TG) are increased to approximately equal concentrations, usually >7.80 mmol/l. So far, there are very few reports on plasma lipid and lipoprotein profiles for type III HLP with apo E2/ 2 phenotype in Japanese population due in part to its rarity [14,15]. We analyzed detailed profiles of serum lipids and lipoproteins in 19 unrelated Japanese type III HLP with apo E2/2 phenotype.
2. Materials and methods
(Daiichi Chemical Tokyo, Japan). Serum TC concentrations z 220 mg/dl is defined as hypercholesterolemia and serum TG concentrations z 150 mg/dl as hypertriglyceridemia [16]. Lipoprotein fractions in sera were separated by serial ultracentrifugation at different densities into VLDL (d < 1.006), IDL (d = 1.006 to 1.019), lowdensity lipoprotein (LDL; d = 1.019 to 1.063) and HDL (d>1.063) according to the method by Havel et al. [17]. The serum remnant-like particle cholesterol (RLPC) concentration was measured using an immunoaffinity mixed gel containing anti-apolipoprotein A-I (apo A-I) and anti-apo B monoclonal antibody, following the method reported by Nakajima et al. [18]. 2.3. Isoform analyzed by isoelectric focusing (IEF) To determine the apo E phenotype for the study subjects, IEF was performed in polyacrylamide gels essentially as described previously [19,20].
2.1. Patients 2.4. Statistical analysis All 19 consecutive type III HLP patients (9 men, 10 women) were Japanese and were recruited from our clinics and affiliated hospitals. Age and body mass index (BMI) of the subjects were 52.5 F 15.9 years old and 23.9 F 2.9 kg/m2, respectively. The diagnosis of type III HLP was performed based on the standard criteria previously reported [15]. Individuals with plasma glucose (PG) z 7.0 mmol/l after overnight fast or those with z 11.1 mmol/l at 2 h after 75-g oral glucose administration documented on >1 occasion were diagnosed as diabetes mellitus (DM). Coronary heart disease (CHD) was diagnosed according to the following criteria: (1) a history of myocardial infarction; (2) a history of angina pectoris together with a positive exercise test result and/or an abnormal coronary angiogram; and (3) a history of coronary artery bypass grafting. Blood sample was obtained after overnight fasting. 2.2. Serum lipoprotein analysis Serum TC, TG and HDL cholesterol (HDL-C) concentrations were determined by standard enzymatic methods. Serum apolipoprotein (apo) concentrations were determined using an immunoturbidimetric assay
All values are expressed as the mean F S.D. StatView-J 5.0 software was used for all statistical analyses. Statistical significance between two groups was determined by unpaired Student’s t test at P < 0.05. The Mann – Whitney test was used to detect the differences between the groups if data did not distribute normally.
3. Results 3.1. Clinical characteristics of the study subjects Among the study subjects, seven patients (36.8%) had obesity and five patients (26.3%) type 2 diabetes mellitus (DM). There were three patients (15.8%) who had coronary heart disease (CHD) and one (5.3%) with atherosclerotic obliterance (ASO). None of the subject had overt cerebral vascular disease. Xanthoma striata palmaris was observed in three patients (15.8%) and Achilles tendon’s xanthoma in three patients (15.8%). Serum lipid, lipoprotein, apolipoprotein, Lp (a), RLP-C concentrations and VLDL cholesterol (VLDL-C)/TG ratio in the study subjects
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are shown in Table 1. Average values for serum TC and TG were 8.1 and 5.2 mmol/l, respectively. All of the patients had hypertriglyceridemia whereas 14 patients (74%) had hypercholesterolemia. Average concentration of HDL-C was within normal limits. Average concentrations of serum apo A-I, A-II and B were within normal limits whereas apo C-II, C-III and E concentrations were markedly increased. In particular, average concentrations for apo E were as high as threefold higher than the upper limits of normal range whereas apo C-II and C-III concentrations were at most twofold higher. 3.2. Apo E/C-III ratio in hyperlipidemic individuals with apo E2/2, E3/2, E3/3, E4/3, E4/2 and E4/4 Fig. 1 shows the comparison of apo E/C-III ratio in individuals with apo E2/2, E3/2, E3/3, E4/3, E4/2 and E4/4. Apo E/C-III ratio from hyperlipidemia with apo E2/2 phenotype was considerably higher than that from other apo E phenotypes, while that from other phenotypes showed almost similar concentrations. Next, we investigated how serum lipids affect this index versus the absolute values of serum apo E concentrations. Apo E values were closely associated with both serum TC and TG concentrations, whereas apo E/C-III ratio had no association with either of these lipids (Table 2). This observation suggests that this index, unlike apo E value, could be a reliable Table 1 Serum lipid, lipoprotein and apolipoproteins values in study subjects (mean F S.D.)
Fig. 1. The comparison of apo E/C-III ratio between hyperlipidemic individuals with various apo E phenotypes. Values are mean F S.D. Hyperlipidemic individuals were used with apo E3/2 (n = 6) [TC 6.70 F 1.35 mmol/l, TG 4.53 F 3.55 mmol/l], E3/3 (n = 19) [TC 6.62 F 1.69 mmol/l, TG 4.27 F 5.84 mmol/l], E4/3 (n = 10) [TC 5.79 F 1.25 mmol/l, TG 5.18 F 3.63 mmol/l], E4/2 (n = 8) [TC 6.73 F 1.17 mmol/l, TG 2.42 F 1.98 mmol/l] and E4/4 (n = 9) [TC 6.70 F 1.38 mmol/l, TG 1.33 F 0.55 mmol/l] besides the study subjects with apo E2/2. ***p < 0.0001 vs. hyperlipidemic individuals with all of the other apo E phenotypes.
marker for screening subjects with apo E2/2 phenotype regardless of serum lipids. To determine the accuracy of apo E/C-III ratio for diagnosing type III HLP, we conducted ROC plot analysis (Table 3). The ROC analysis showed that sensitivities almost equaled specificities at apo E/C-III between 0.70 and 0.75 for identifying type III HLP with apo E2/2. 3.3. Gender differences in the parameters on lipoprotein metabolism between men and women
Normal range N Age, year BMI, kg/m2 TC, mmol/l TG, mmol/l HDL-C, mmol/l Apo A-I, mg/dl Apo A-II, mg/dl Apo B, mg/dl Apo C-II, mg/dl Apo C-III, mg/dl Apo E, mg/dl Apo E/C-III, mg/dl Lp(a), mg/dl RLP-C, mg/dl VLDL-C/TG
19 52.5 F 15.9 23.9 F 2.9 8.1 F 3.2 5.2 F 2.9 1.1 F 0.2 133 F 24.7 37.5 F 8.0 120 F 63.8 11.1 F 4.9 20.4 F 9.3 18.6 F 7.5 0.911 9.9 F 5.0 n = 7 19.6 F 8.1 n = 7 0.32 F 0.07
We also analyzed the differences in the parameters on lipoprotein metabolism between men and 3.4 – 5.7 0.4 – 1.7 0.9 – 2.2 97 – 167 24 – 47 52 – 130 1.5 – 5.1 4.7 – 11.9 2.1 – 6.0 34.6> 7.5>
Table 2 Relationship of TC or TG with apo E or E/C-III R
p
To TC Apo E 0.862 < 0.0001 Apo E/C-III 0.091 NS - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - -- - - - - To TG Apo E 0.828 < 0.0001 Apo E/C-III 0.125 NS TC, total cholesterol; TG, triglycerides.
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Table 3 ROC analysis: ranges of cutoff lines and their corresponding specificities and sensitivities for apo E/C-III
RLP-C concentrations and VLDL-C/TG ratio (data not shown).
Cutoff line
Specificities
1-Specificities
Sensitivities
1.55 1.50 1.45 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05 1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25
1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.96 0.96 0.96 0.96 0.96 0.96 0.92 0.92 0.88 0.85 0.85 0.77 0.67 0.58 0.40 0.31 0.15 0.06 0.02 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.04 0.04 0.04 0.04 0.04 0.04 0.08 0.08 0.12 0.15 0.15 0.23 0.33 0.42 0.60 0.69 0.85 0.94 0.98 1.00
0.00 0.11 0.11 0.11 0.11 0.11 0.22 0.28 0.33 0.33 0.33 0.39 0.50 0.56 0.61 0.67 0.83 0.89 0.94 0.94 0.94 0.94 0.94 1.00 1.00 1.00 1.00
3.4. Profiles of lipoprotein fractions analyzed by ultracentrifugation from the study subjects Table 4 shows the profiles of lipoprotein fractions analyzed by ultracentrifugation from the study subjects. VLDL-C concentrations were considerably increased. IDL cholesterol (IDL-C) concentrations were also increased whereas LDL cholesterol (LDL-C) concentrations were low. VLDL-TG, IDL TG, LDL-TG and HDL-TG concentrations were increased. HDL-C was within normal limits while HDL-TG concentrations were increased in type III HLP. Again, there was no significant difference between men and women for the profiles of lipoprotein fractions. The VLDL-C/TG ratio, which is regarded as a useful index for diagnosing type III HLP, was 0.32 F 0.07 in our study subjects.
4. Discussion
women. There was no statistically significant gender differences for TC, TG, HDL-C, apo A-I, apo A-II, apo B, apo C-II, apo C-III, apo E, Lp(a),
The main findings of the present study on detailed analysis of serum lipids and lipoproteins from 19 unrelated Japanese type III HLP with apo E2/2 phenotype are as follows: (1) all of the study subjects had increased serum TG, while 74% of the subjects had hypercholesterolemia; and (2) the increase of apo E concentration was far more pronounced than that of
Table 4 Ultracentrifugation analysis of serum lipoprotein values in men and women (mean F S.D.)
N Cholesterol VLDL IDL LDL HDL Triglycerides VLDL IDL LDL HDL
Total
Men
Women
Normal controls
19 7.8 F 3.1*** 3.7 F 2.1*** 1.1 F 0.5** 1.4 F 0.8*** 1.1 F 0.3 4.5 F 2.5*** 3.3 F 1.9*** 0.4 F 0.2*** 0.3 F 0.1** 0.3 F 0.1***
9 7.1 F 2.5*** 3.6 F 2.3*** 0.9 F 0.4*** 1.4 F 0.9*** 0.9 F 0.3* 4.5 F 2.6*** 3.6 F 2.4*** 0.3 F 0.2*** 0.3 F 0.1* 0.3 F 0.1***
10 8.5 F 3.6*** 3.8 F 2.0*** 1.2 F 0.5*** 1.4 F 0.7*** 1.2 F 0.3 4.5 F 2.5*** 3.0 F 1.3*** 0.4 F 0.3*** 0.3 F 0.1* 0.3 F 0.03***
20 4.8 F 0.7 0.4 F 0.2 0.2 F 0.1 2.5 F 0.6 1.2 F 0.3 1.0 F 0.4 0.5 F 0.3 0.1 F 0.03 0.2 F 0.1 0.2 F 0.03
VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. * p < 0.05 vs. control. ** p < 0.01 vs. control. *** p < 0.001 vs. control.
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apo C-III, causing the ratio of apo E/C-III to be considerably higher than individuals with other apo E phenotype. The findings that average concentrations for serum TC and TG in our study subjects were lower than those from type III HLP in western countries were in line with the observation in other Japanese group [15]. It is known that hyperlipidemia with apo E2/2 has marked increase of apo E concentrations [21 –25]. However, we presume that high serum apo E concentration per se is not sufficient to segregate apo E2/2 subjects in view of the fact that apo E concentration depends highly on serum TC and TG concentrations (Table 2). Indeed, in the U.S., the proposed diagnostic concentrations of plasma apo E concentrations for type III HLP is controversial [21,24]. In contrast, the ratio of apo E/ C-III is not affected by TC and TG concentrations, which is close to 0.9 in individuals with apo E2/2 (Fig. 1; Table 2). Thus, we suggest that this index is a useful marker for screening type III HLP with apo E2/2 phenotype from other type of HLP. We assume it is possible to segregate type III HLP with apo E2/2 regardless of the degree of hyperlipidemia. Another marker for diagnosing type III HLP might be a VLDLC/TG ratio of >0.30 [9]. In our study, the average value for this ratio was 0.32 F 0.07 which is close to the report by Janus et al. [23] and is higher than the finding by another Japanese group [15]. Several other markers have been shown useful for segregating HLP with apo E2/2 phenotypes [26,27]. Sakai et al. [26] showed that apo B-48/TG was almost equal to 0.15 in HLP with apo E2/2 phenotypes, whereas in all of other lipid phenotype, this ratio was V 0.064. Wang et al. [27] proposed that RLP-C/TG is a useful marker for segregating type III HLP from other type of HLP. We presume that these markers are also very useful for the diagnosis of type III HLP. However, a big advantage of our marker over these reports is that we do not have to use values such as apo B48 and RLP-C which are not routine available. The ultracentrifugation analysis of serum lipoproteins showed that the HDL-C was within normal limits while HDL-TG concentrations were increased in type III HLP, suggesting that HDL particles are relatively TG-rich in type III HLP. Subjects with type III HLP occasionally have xanthoma striata palmaris and Achilles tendon’s xanthoma, which were found 15.8% and 15.8%, respectively, in our study subjects. One of the clinically most
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important complications of type III HLP is known to be coronary heart disease (CHD) which was shown in 15.8% of our study subjects, whereas other Japanese group has reported its frequency to be 37.5%. The precise factors causing this discrepancy in CHD frequency between our study and other group remains to be clarified; but it may be related to the fact that in the present study, 37% of the patients were obese and 26% had type 2 diabetes, while their study subjects consisted in 75% obesity and 43.5% DM. In conclusion, we conducted a detailed analysis of serum lipids and lipoproteins in Japanese type III HLP with apo E 2/2 and therefore propose that apo E/C-III is a useful index for screening HLP with apo E 2/2 phenotype. Acknowledgements We thank Ms. Yoshida and Ms. Mizuno for their excellent technique for analyzing serum lipoprotein profiles of the study subjects. References [1] Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988;240: 622 – 30. [2] Mahley RW, Rall Jr SC. Apolipoprotein E: far more than a lipid transporter protein. Annu Rev Genomics Hum Genet 2000;01:507 – 37. [3] Mahley RW, Rall Jr SC. Type III hyperlipoproteinemia: the role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B, editors. The metabolic and molecular bases of inherited disease, vol. 2. New York: McGraw-Hill; 2001. p. 2835 – 62. [4] Weisgraber KH, Innerarity TL, Mahley RW. Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine – arginine interchange at a single site. J Biol Chem 1982;257:2518 – 21. [5] Utermann G, Pruin N, Steinmetz A. Polymorphism of apolipoprotein E: III. Effect of a single polymorphic gene locus on plasma lipid concentrations in man. Clin Genet 1979;15: 63 – 72. [6] Utermann G, Vogelberg KH, Steinmetz A, Schoenborn W, Pruin N, Jaeschke M, et al. Polymorphism of apolipoprotein E: II. Genetics of hyperlipoproteinemia type III. Clin Genet 1979;15:37 – 62. [7] Utermann G, Hees M, Steinmetz A. Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinaemia in man. Nature 1977;269:604 – 7.
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